Native human cystatin C is known (see ref. 1). It consists, in its naturally occuring form isolated from body fluids, of a mixture of two types of polypeptide, differing in that type I has the N-terminal amino acid sequence: Ser-Ser-OH-Pro-Gly-while type II, called 3-des-OH-cystatin C, has the following N-terminal sequence: Ser-Ser-Pro-Gly-. Thus, the two types only differ in the third amino acid residue. Cystatin C has a useful biological activity as explained in the following. Depending on the origin of the preparations and the extraction method, the activity may vary somewhat.
Cystatin C has 120 amino acids. The amino acid sequence of recombinant 3-des-OH-Cystatin C is as follows Ser-Ser-Pro-Gly-Lys-Pro-Pro-Arg-Leu-Val-Gly-Gly-Pro-Met -Asp-Ala-Ser-Val-Glu-Glu-Glu-Gly-Val-Arg-Arg-Ala-Leu-Asp -Phe-Ala-Val-Gly-Glu-Tyr-Asn-Lys-Ala-Ser-Asn-Asp-Met-Tyr -His-Ser-Arg-Ala-Leu-Gln-Val-Val-Arg-Ala-Arg-Lys-Gln-Ile -Val-Ala-Gly-Val-Asn-Tyr-Phe-Leu-Asp-Val-Glu-Leu-Gly-Arg -Thr-Thr-Cys-Thr-Lys-Thr-Gln-Pro-Asn-Leu-Asp-Asn-Cys-Pro -Phe-His-Asp-Gln-Pro-His-Leu-Lys-Arg-Lys-Ala-Phe-Cys-Ser -Phe-Gln-Ile-Tyr-Ala-Val-Pro-Trp-Gln-Gly-Thr-Met-Thr-Leu -Ser-Lys-Ser-Thr-Cys-Gln-Asp-Ala. Native human cystatin C has been isolated from urine and its amino acid sequence has been determined (see ref 1), after fragmentation of the polypeptide chain, partly using cyanogen bromide and partly using trypsin cleavage. During the analysis it was shown that the amino acid in position 3 (Pro) was hydroxylated to a degree of about 50% but somewhat varied. Cystatin C, in its native form, thus consists of a mixture of two closely related components of almost the same size, the first of which is hydroxylated in position 3 (Pro) while the second (3-des-OH-cystatin C) is not. This fact has not previously been recognized as important and cystatin C has therefore been used and studied in the native form as a mixture.
Cystatin C has now, according to the invention, been separated into the two components and it has surprisingly been shown that the biological effect is solely connected with 3-des-OH-cystatin C.
As is known from literature, cystatin C is an efficient inhibitor for cystein-proteinase, such as papain and cathepsin B. Native human cystatin C has been described by Grubb et al. (ref. 1) who has indicated the amino acid sequence and also described some biological properties.
Cystein-proteinases participate in the intracellular catabolism of proteins and peptides, in the proteolytical conversion of prohormones, in the extracellular degradation of collagen and in the penetration of normal tissue with malignant cells.
As cystatin C and thus also 3-des-OH-cystatin C inhibits cystein-proteinases which accelerate cancer growth or metastasis-formation, it is a potential cancer-inhibiting agent.
Furthermore, cystatin C and thus also 3-des-OH-cystatin C possess antiviral activity. Thus, it is highly effective against herpes simplex virus. In this respect it has a higher activity than the known compound N-benzyloxycarbonyl-leucyl-valyl-glycine diazomethyl ketone (Z-LVG-CHN.sub.2 and Acylovir). Preparations containing cysterin C may thus be used in the treatment of herpes simplex.
Cystein-proteinases, e.g. chymopapain, may also be used for treatment of slipped disc. If the cystein-proteinases diffuse into the cerebrospinal fluid, cerebral hemorrhage will occur as a serious side effect of the therapeutic treatment of slipped disc. As cystatin C and thus also 3-des-OH-cystatin C inhibit such side-effect, it has therapeutic use in this connection.
Of specific important is the activity of cystatin C to counteract hereditary cerebral hemorrhage with amyloidosis. In some cases of this disease there has been found deposits of a so-called HCHWA amyloid protein (Refs. 7 and 8). This protein has been shown to be a variant of cystatin C as it lacks the first 10 amino acids of the cystatin C sequence while the amino acid Leu in position 68 of cystatin C, corresponding to position 58 of the HCHWA protein is replaced by the amino acid Glu. It is assumed that this variant of cystatin C has connection to or is responsible for the disease. If this is the case, it may be expected that 3-des-OH-cystatin C would be useful in the treatment of the disease. In the decomposition of collagen in tissue cystein-proteinases participate, and 3-des-OH-cystatin C may therefore probably inhibit such decomposition of tissue.
The cystatin C amino acid sequence isolated from human urine has been characterized and appears to be somewhat homologous to c-Ha-ras oncogene products.